ABSTRACT The antiviral innate immune response is the first line of defense against viral infection. Antiviral immunity is initiated by cellular proteins that sense the presence of virus inside an infected cell. As a result, this signaling cascade culminates in the production of type I interferons (IFN) and hundreds of IFN-stimulated genes (ISGs). IFN-responsive RNA binding proteins that either inhibit RNA virus replication or have m6A-selective recognition of cellular RNAs have been described previously; however, if selective binding of N6-methyladenosine (m6A) is required for the function of these proteins during an antiviral response is not clear. Our preliminary studies have identified an ISG that preferentially binds to m6A-containing RNAs and negatively regulates RNA virus infection. PAR-CLIP (photoactivatable crosslinking and immunoprecipitation coupled to next generation sequencing) analysis reveals that it binds to cellular RNAs in regions known to contain m6A. However, the impact of m6A-selectivity on the antiviral innate immune effector mechanisms of this ISG is unclear. Therefore, the goal of this proposal is to define the biochemical interactions of one human ISG that acts during the IFN response to recognize m6A-modified nucleotides on viral RNA. Based on our preliminary data, the central hypothesis of this proposal is that this ISG binds to RNAs in an m6A-dependent manner to regulate the host response to viral infection. Guided by our preliminary data, this hypothesis will be tested by the following two specific aims: 1) Define the mechanisms of RNA recognition and m6A discrimination of this IFN-stimulated m6A-reader; 2) Determine the role RNA recognition by this IFN-stimulated m6A-reader in restricting RNA virus infection. In Aim 1, the molecular basis for recognition of m6A-containing RNAs by this ISG will be determined. In Aim 2, how RNA-binding and m6A-recognition of this protein define the outcome of viral infection will be explored in human cells infected with RNA viruses of global public health concern. Taken together, the work proposed in this application will be significant and innovative because it will define a new role for ISGs in recognizing m6A-modified RNA to regulate the antiviral innate immune system and viral infection. This work will inform therapeutics and treatment strategies for RNA virus infection and autoimmune diseases. Further, an increased understanding of how viral infection is controlled by innate immune effectors will have implications for therapeutics and vaccine responses to limit the burden of RNA virus infection.